Recent clinical gene therapy trials for immunodeficiency disorders have utilized integrating retroviral vectors to provide stable transmission of various therapeutic genes into hematopoietic stem cells. While these trials have demonstrated proof of efficacy in severe combined immunodeficiencies and Wiscott-Aldrich syndrome, there have been six cases of Tcell malignancies due to vector-induced activation of cellular proto-oncogenes, most notably LMO2. In most if not all of these cases, the interaction of vector-encoded enhancers, located in the viral long terminal repeats, with adjacent cellular proto-oncogene promoters resulted in inadvertent gene activation events and transformation. A major goal in the field of gene therapy is to eliminate or reduce the incidence of this serious side effect through the elimination of enhancers and other viral transcriptional elements, the use of self-inactivating lentiviral vectors, and the use of chromatin insulator elements that contain enhancer blocking sequences. This application focuses on identifying new, more effective enhancer blocking elements that would provide maximum activity in human T cells and therefore allow safer gene therapy vectors for T cell immunodeficiency disorders. The focus of specific aim 1 is to develop a sensitive and relevant cellular assay for screening libraries of potential insulator elements; in specific aim 2, the newly developed insulator screening system will be used to identify novel enhancer blocking elements with high level activity in human T lymphocytes. An important current obstacle for the field is the relative lack of effective insulator elements for this purpose; much of the field uses an element from the chicken -globin locus (cHS4) that is relatively tissue specific and only partially active in T cells. We will build upon a novel system developed here at St. Jude for homologous gene targeting in Jurkat T cells to screen a library of CTCF binding sequences derived from T cells for enhancer blocking activity. Our goal is to identify new blocking elements that are highly active in T cells.